In regulated rivers, dams alter longitudinal gradients in flow regimes, geomorphology, water quality and temperature with associated impacts on aquatic biota. Unregulated tributaries can increase biodiversity in regulated environments by contributing colonists to the main channel and creating transitional habitats at a stream junction. We assessed whether unregulated tributaries influence macroinvertebrate communities in two mainstem rivers during summer low-flows. Three tributary junctions of upland cobble-gravel bed streams were surveyed in an unregulated and a regulated river in the Sierra Nevada Mountains, California, USA. We found distinct physical habitat conditions and increased macroinvertebrate abundance and diversity in unregulated tributaries on the regulated river, but macroinvertebrate diversity did not increase downstream of tributary junctions as predicted. On the unregulated river, macroinvertebrate diversity was similar in upstream, downstream and unregulated tributary sites. Our findings highlight that unregulated tributaries support high macroinvertebrate diversity and heterogeneous communities compared to the mainstem sites in a regulated river, and thus likely support ecological processes, such as spill-over predation, breeding and refugia use for mobile taxa. We suggest unregulated tributaries are an integral component of river networks, serving as valuable links in the landscape for enhancing biodiversity, and should be protected in conservation and management plans.